The antibacterial activity of methanolic Anacyclus pyrethrum and Pistacia lentiscus L. extract on Escherichia coli.

BACKGROUND AND OBJECTIVES
Antibiotic therapy is the main choice in treatment of Escherichia coli induced infections. Using herbal medication is an alternative choice in treatment of diseases. The aim of this study was to determine the antibacterial activity of two traditionally used herbs in Iranian medicine, Anacyclus pyrethrum and Pistacia lentiscus L., on Escherichia coli.


MATERIALS AND METHODS
The antibacterial effect of methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L. were examined in disk diffusion and skipped wells methods by measuring the diameter of inhibition zones around wells containing different concentrations of extracts from (10-1000 mg/ml) using standard broth macrodilution, method the MIC and MBC were defined.


RESULTS
The methanolic extract of Anacyclus pyrethrum from 300 to 1000 mg/ml and the methanolic extract of Pistacia lentiscus L. from 30 to 1000 mg/ml showed antibacterial activity on Escherichia coli. The MIC of Anacyclus pyrethrum and Pistacia lentiscus L. methanolic based extract were 800 and 1000 mg/ml, respectively. The MBC was achieved at 800 mg/ml for methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L.


CONCLUSION
The methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L. have antibacterial effect on Escherichia coli bacteria. This activity is dose-dependent.


INTRODUCTION
Escherichia coli is a facultative Gram negative bacterium that is generally found in the lower intestine (1). The virulent strains of E. coli cause gastroenteritis and infections of urinary tract. In addition to fluid and electrolytes replacement, the antibiotic therapy is the main choice in treatment of diseases caused by this organism (2)(3).
Antibiotic therapy is facing with different problems ranging from hypersensivity to bacterial resistance. Herbal medication is an alternative choice in treatment of different diseases. The native herbal -based medicine are easy available and non-expensive. The effectiveness of some native herbal -based medicine on E. coli has been shown in Africa, England and China (4)(5)(6)(7).
Anacyclus pyrethrum (locally known as akarkara) and Pistacia lentiscus L. (locally named mastaki) are traditionally used herbs in Iranian medicine by physicians such as Avicenna (8). Anacyclus pyrethrum (pellitory) from Anacyclus genus is a native plant of India and Arabic countries. It has different therapeutic effects such as antmicrobial, analgesic and antioxidant activitivities (9)(10). It has been shown that Anacyclus pyrethrum has antimicrobial effects on Candida albicans, Staphylococcus aureus and strong larvicidal activity against malaria (11)(12).
Pistacia lentiscus L. is a native herb of Africa and Mediterranean countries with antimicrobial, antifungal, antioxidant and anti-inflammatory activities (13)(14). It has good effect on gastrointestinal diseases due to its anti Helicobacter pylori activity (14)(15).
The aim of this study was to determine the antibacterial activity of Anacyclus pyrethrum and Pistacia lentiscus L. on E. coli. Based on our knowledge this is the first study on the antibacterial activity of methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L. on this organism

MATERIALS AND METHODS
The antibacterial activites of methalonic extracts of Anacyclus pyrethrum and Pistacia lentiscus L. on E. coli were examined using well diffusion method. The Anacyclus pyrethrum and Pistacia lentiscus L. were purchased from traditional pharmacies in Shiraz, Iran. The samples were verified by the Department of Pharmacognosy, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Extraction. 200ml of 95% methanol was added to 200g of the chopped, powdered roots of Anacyclus pyrethrum and Pistacia lentiscus L. in a sterile flask. The mixture left for 24 hours at room temperature and filtered with No.1 filter paper (Whatman Co Germany.) with 150µm diameter for liquor filtration. The extract was dried in Water bath at 70 Cº for one week. Dried powdered extract was kept at 4°C at tightly closed vial. Different concentrations from 10 mg/ml to 1200 mg/ml in distilled water were prepared from stock solution (16).
Organism. Escherichia coli (ATCC 25922) was obtained from the bacterial collection of Department of Microbiology, Medical Faculty, Shahed University.

Disk diffusion test.
A swab of bacterial suspension was streaked on Muller Hinton agar plates (Liofilchem Company, Italy).
The columns adjusted to 1.5× 10 8 bacterial/ml and used sterile blank disks of extract dilution (McFarland 0.5 turbidity standard). After overnight 37°C aerobically condition incubation, the zones of inhibition were measured by using an Antibiotic disk Zone Reader after 24 h.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
The MIC was measured by broth Macro dilution test (tube Dilution) method updated by Clinical and Laboratory Standards Institute (17). The lowest concentration of extract that inhibited the visible growth of organism was recorded as MIC. 10 µl of bacterial suspensions with turbidity of McFarland 0.5 were added to test tubes. 1ml of incubated solution with determined MIC were cultured and kept incubated at 37°C for 24 h. The antimicrobial extracts concentration that show inhibition (>MIC) may have killed the bacterium. Subculture plate less than 0.1% of the initial inoculum, first concentration without bacteria growth was considered as the MBC.

Anacyclus pyrethrum. The methanolic extract of
Anacyclus pyretprum had not inhibitory effect on E. coli at 10 to 200 mg/ml concentrations. From 300 to 1000 mg/ml the inhibition zone was noted. Table 1. shows the inhibition zone diameters in different concentrations of methanolic extract of Anacyclus pyrethrum on E. coli.
The MIC of methanolic based extract of Anacyclus pyrethrum was 800mg/ml ( Table 2).
The MBC of methanolic extract of Anacyclus pyrethrum was achieved at 900 mg/ml.
Pistacia lentiscus L. The methanolic extract of Pistacia lentiscus L. had not inhibitory effect on E. coli at 10 to20 mg/ml concentrations. From 30 to 1000 mg/ml the inhibition zone was noted. Table 1 shows the inhibition zone diameters (mm) in different concentrations of methanolic extract of Pistacia lentiscus L. on E. coli.
The MIC of methanolic based extract of Pistacia lentiscus L. was 1000mg/ml. (Table 2) The MBC of methanolic extract of Pistacia len-tiscusL. was at 1000 mg/ml.

DISCUSSION
The study shows methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L. have antibacterial effect on E. coli. This activity is dose-dependent.
Some previous researches have shown the effectiveness of native derived herbal -based medicine on E. coli such as the mixture of combining Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus from Africa and Potentilla reptans L. from Anglo-Saxon native herbs (4)(5).
The study shows the methanolic extract of Anacyclus pyrethrum from 300 to 1000 mg/ml had inhibitory effect on E. coli. Based on our knowledge this is the first study on the antibacterial activity of methanolic extract of Anacyclus pyrethrum on E. coli.
Selles et al. showed that the essential oil from Algerian Anacyclus pyrethrum L. has activity against Candida albicans and Staphylococcus aureus. Oxygenated sesquiterpenes was the main effective substance (12).
Studies on biologically active substance of Anacyclus pyrethrum and its antibacterial effects are very rare. It has not been elucidated that which active component or either biologic mechanism of Anacyclus pyrethrum has antibacterial activity on E. coli.
It seems that different components of Pistacia lentiscus L. have different effects on different bacteria. Mharti et al. demonstrated that germanicol, thunbergol, himachalene, trans-squalene, terpinyl propionate, 3,3-dimenthol and cadina-1.4-diene derived from essential oil of the leaves of Pistacia lentiscus L. have antibacterial effect on Klebsiella pneumoniae, but not on Pseudomonas aeruginosa (23).
The present study shows that methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L. have antibacterial effect on E. coli. More researches on identifying the effective components of Anacyclus pyrethrum and Pistacia lentiscus L. on Escherichia coli are necessary.
In conclusion, the methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L. have antibacterial effect on Escherichia coli bacteria. This activity is dose-dependent.